The invention relates to protease inhibitor compounds useful in treatments of neurodegenerative, cardiovascular, pulmonary, neuromuscular, musculovascular and autoimmune diseases characterized by tissue damage, induction of apoptosis, cell death and/or the accumulation of amyloid proteins in deposits, plaques, fibrils and tangles.
Cysteinyl proteases are implicated in important cellular processes including endocytosis and degradation of proteins, immune recognition, apoptosis and processing of amyloid precursor protein. Cysteinyl proteases are localized in the endosomal lysosomal compartment, possibly the endoplasmic reticulum and cytosol. While many of the molecular details are at present uncertain, the importance of cysteinyl proteases at the cellular level and at an organism level cannot be overstated.
In Alzheimer""s disease (AD), amyloid precursor protein (APP; i.e., APP695) is proteolytically processed in neural cells and Axcex2 cleavage fragments are secreted into cerebral extracellular perivascular matix. Plaques containing Axcex2 act as a pathological landmark of AD (2-5) and in vitro studies have shown to that Axcex2 can aggregate into insoluble fibrils which ultrastructurally resemble amyloid plaques (7). Aggregated in a fibrillar beta-pleated sheet configuration, Axcex2 is thought to form a nidus for binding other proteins contributing to Alzheimer""s dementia and pathology. Axcex2 fibrils are reportedly toxic for cultured neurons in vitro (8), possibly through induction of apoptosis (9), initiation of free radical formation (10) or increases in peroxide (H2O2) levels in cells (11).
APP, synthesized by neuronal cells, full length APP may be degraded in lysosomes (21,22), resulting in non-amyloidogenic fragments, or it can be proteolytically processed to polypeptides of 110 kDa, i.e., sAPP (16-17); 9-10 kDa (17); and 4.2 kDa, i.e., Axcex2 (18-19). Characterization of fragments and protease inhibitor profiles suggest three intracellular enzymes, termed secretases, differing as follows: namely,
Alpha-secretase (xcex1-secretase) mediates cleavage of APP695 and APP751 producing sAPP (110 kDa) and a 9-10 kDa carboxy-terminal-fragment (CTF; i.e., APP611-695). The CTF fragment may undergo further degradation in lysosomes (17). Products of lysosomal and xcex1-secretase cleavages are generally considered to be non-amyloidogenic;
Beta-secretase (xcex2-secretase) mediates cleavage of APP695 in the carboxy-terminal region (i.e., at APP Met595-Asp596; Axcex2 Asp1; 23), producing a 11.4-11.8 kDa fragment, 100 amino acids in length (CTF100), and containing the Axcex2 domain; and,
Gamma-secretase (xcex3-secretase) mediates cleavage in the C-terminal transmembrane domain of APP, and possibly also CTF100, to release the Axcex21-39, Axcex21-40 and/or Axcex21-42 peptides. Both Axcex21-40 and Axcex21-42 are reportedly amyloidogenic; with Axcex21-42 being more active than Axcex21-40 (7,24,25).
Sensitivity of secretases to protease inhibitors has been studied in cell-based assays, i.e., by treating APP producer cells with protease inhibitors. Results have varied in different cells and under differing conditions of culture. In certain reports, APP cleavage is reportedly inhibited by serine protease inhibitors; in others by inhibitors of chymotrypsin-like enzymes; and in others by inhibitors of cysteinyl proteases such as cathepsins B, D, H, L and S and calpain. Studies with leupeptin (26,27), E64 and Z-Phe-Ala-CHN2 (28), i.e., lysosomal enzyme inhibitors, have failed to implicate known lysosomal hydrolases in production of Axcex2. Similarly, although calpain inhibitor I, a caspase inhibitor, was initially reported effective in reducing Axcex2 levels in vitro (29), subsequent findings seem at odds with an effect on a putative xcex3-secretase (30). Dipeptidyl-aldehyde inhibitor, Z-L-Val-L/D-Phe-aldehyde (MDL 28170) and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] were also once thought to inhibit Axcex21-42 production (32). Since non-specific acidotropic agents such as NH4Cl and chloroquine, or Golgi inhibitors such as brefeldin A and monensin (19,27), reportedly affect APP processing it is possible that it takes place in the endoplasmic reticulum (ER) and/or Golgi apparatus. Specific and targeted inhibitors of secretase enzymes have (to date) proven elusive.
In apoptosis, an intracellular cascade of cysteinyl proteases, termed caspases, is activated by radiation damage to cellular DNA or structures (e.g., mitochondria), or by removal of one or more critical growth factors (e.g., EGF, PGF, TGFxcex2, NGF, BDNF), glucose or serum. In addition, a variety of different receptor ligand interactions at the cell surface may trigger apoptosis in vitro: e.g., anti-Fas antibody or Fas ligand binding to Fas receptor; IL-1 binding to IL-1Rxcex2; TNFxcex1 binding to TNFR1 and TNFR2; Apo 3 ligand binding to Death Receptor 3; glucocorticoids binding to cell surface glycocorticoid receptors; TRAIL, TRANCE and NOC18 binding to yet other cell surface xe2x80x9cdeathxe2x80x9d receptors. Important cysteinyl proteases activated in this manner are thought to include caspases 3, 8 and 9.
Apoptosis has recently been implicated in mechanisms by which tissue damage is triggered following traumatic injury, vascular insufficiency and stroke.
Using knockout mice, Cathepsin S is reportedly involved in class I MHC antigen processing, i.e., of self and viral antigens, while Cathepsin L is involved in class II processing of foreign proteins.
Thus, protease inhibitors find a variety of important medical and veterinary uses in treatments of diseases, as well as, uses in the food, dairy, agriculture, chemical and biotechnology industries.
Cysteinyl protease inhibitors, pharmaceutical compositions and methods of treating protein processing disorders, trauma, stroke, autoimmunity, neurodegenerative and cognitive disorders including Alzheimer""s disease, are disclosed. The di- and tri-peptide analogues with unnatural xcex1-amino side chain residues and derivatized amino and carboxyl terminal residues constitute highly effective inhibitors of cysteinyl proteases including e.g. cathepsins, caspases, calpains and secretases. Methods and pharmaceutical compositions are provided for modulating activities of cysteinyl proteases involved in diseases including methods for altering secretase-mediated processing of APP in Alzheimer""s disease and aberrant cathepsin S-mediated processing of self proteins for MHC class I presentation in autoimmune diseases. The instant compounds include vinyl sulfides, sulfoxides, sulfones, amines, amine oxides, phosphines, phosphine oxides, phosphinates and phosphonates of peptidyl analogs of di- and tri-peptides, where the vinyl substituent in the peptidyl analog preferably occupies the carboxyl-terminal position and consists of a substituted alkenyl or alkynyl side chain.
Embodiments of the invention provide cysteinyl protease inhibitors. The instant compounds are vinyl heterocyclic alkyls and aryl adducts of peptidyl analogs of natural xcex1-amino acid containing cysteinyl protease substrates; according to the following FORMULA I, II or III: namely, 
wherein R1 is an unnatural amino acid side chain, preferably a lower alkenyl or lower alkynyl that is unsubstituted, or alternatively, substituted with W,
W, in turn, is halo, hydroxy, alkyl, aryl, heterocycle, heteroaryl, alkoxy, aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, heterocycle-carbonyl, nitro, haloalkyl; preferably, trifluoromethyl, cyano, amino or aminocarbonyl; and most preferably, a side chain not present in a natural amino acid;
X is xe2x80x94CHxe2x95x90CHxe2x80x94(CHxe2x95x90CH)kxe2x80x94(CH2)j or xe2x80x94CH2CH2xe2x80x94(CH2)b where xe2x80x9ckxe2x80x9d is 0-9, preferably 0-3, and most preferably 0; xe2x80x9cjxe2x80x9d is 0-4, preferably 0-2, most preferably 0; and, xe2x80x9cbxe2x80x9d is 0-6, preferably 3-6, most preferably 3 or 4;
Y is S, SO, SO2, NR2O, xe2x80x94N(O)(R20)xe2x80x94, xe2x80x94PR20, xe2x80x94P(O)(R20)xe2x80x94, xe2x80x94P(O)Oxe2x80x94 or xe2x80x94P(O)(OR20)Oxe2x80x94; wherein, R20 is hydrogen, alkyl or aryl;
Z is xe2x80x94(CH2)ixe2x80x94A; xe2x80x9cixe2x80x9d is 0-4, preferably 0-2, most preferably 0; wherein, A is a substituted or unsubstituted heterocycle, heteroaryl or aryl; where the substituents consist of one or more L groups; wherein, L is halogen, lower alkyl, lower alkenyl, lower alkynyl, alkoxy, aryloxy, hydroxy, haloalkyl, preferably trifluoromethyl, nitro, nitrile, alkylthio, phenyl, and xe2x80x94NR30R31; and, in optional embodiments L may be further substituted with W, as set forth above, but preferably L is selected from the substituents set forth in regard to V, below;
R30 and R31 are each independently H, alkyl, preferably lower alkyl, hydroxy or halo lower alkyl, and most preferably trifluoromethyl;
V is OH, halogen, lower alkyl, preferably methyl or ethyl or halogen-substituted lower alkyl, preferably halogen-substituted methyl or ethyl, and is more preferably OH;
R2, R3, R4, R5, R6, R7, R8, Q and n are each selected from (i), (ii), (iii), (iv), (v), (vi) and (vii) as follows;
(i) R3 and R5 are each independently selected from a side chain of a naturally occurring xcex1-amino acid; H; alkyl, preferably lower (C1-6) alkyl; alkenyl, preferably C2-10 alkenyl; alkynyl, preferably C2-6 alkynyl; aryl, aralkyl, aralkenyl, aralkynyl; heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl; L-substituted aryl, L-substituted aralkyl, L-substituted aralkenyl, L-substituted aralkynyl; M-substituted heteroaryl, M-substituted heteroaralkyl, M-substituted heteroaralkenyl and M-substituted heteroaralkynyl; wherein,
M is lower alkyl, preferably C1-4 alkyl, or alternatively, M is halo-lower alkyl, preferably C1-4 haloalkyl, but most preferably M is trifluoromethyl;
R2, R4, R6, and R8 are each independently selected from among H; lower alkyl, preferably C1-4 alkyl; cycloalkyl, preferably lower cycloalkyl; and cycloalkylalkyl, preferably lower cycloalkylalkyl;
R7 is selected from among C1-6 alkyl; RARBCH; L-substituted or unsubstituted aryl; alkenyl; alkynyl; 9-fluorenyl; aralkyl; aralkenyl; aralkynyl; L-substituted or unsubstituted monocylic or bicyclic heterocycle; L-substituted or unsubstituted monocyclic or bicyclic aryl, L-substituted or unsubstituted bicyclic aryl-heteroaryl, and L-substituted or unsubstituted monocyclic or bicyclic heteroaryl;
Q is xe2x80x94C(O)xe2x80x94, xe2x80x94Oxe2x80x94C(O)xe2x80x94, xe2x80x94S(O)2xe2x80x94 or xe2x80x94HNxe2x80x94C(O)xe2x80x94;
n is zero or one;
RA is xe2x80x94(T)m, xe2x80x94(D)m, xe2x80x94R3, wherein T is 0 or NH, D is C1-4 alkyl or C2-4 alkenyl and m is zero or one; and
RB is selected in the same manner as in regard to R3 and R5, above; or
(ii) R2, R5 and R8 are selected as in (i), above;
R3 and R4 are selected as in either of (i), above, or (iv), below;
n is zero; and
R6 and R7 together with the atoms to which each is attached form a 3-10 membered ring, preferably a 4-8 membered ring, most preferably a substituted or unsubstituted 5-6 membered heterocyclic moiety containing 1-5 heteroatoms, where the substituents of the ring are selected either from those identified in regard to L, above, but, preferably those identified in regard to V, above, and most preferably the substituents so chosen will, together with the ring members will form a morpholino, thiomorpholino, pyrrolidinyl, V-substituted pyrrolidinyl, or 4-hydroxypyrrolidinyl ring; or
(iii) R2, R3, R4, R5 and R8 are selected as set forth in regard to (i), above;
Q is xe2x80x94C(O)xe2x80x94
n is one; and
R6 and R7 are substituted or unsubstituted carbonyl (xe2x80x94(Cxe2x95x90O)xe2x80x94), phenyl, a heteroatom, lower alkylene, preferably C2-3 alkylene, or lower alkylene linked to a heteroatom, preferably C2-3 alkylene linked to a heteroatom; together with the atoms to which they are attached R6 and R7 form a cyclic moiety, preferably a 4-6 membered cyclic or 8-12 membered bicyclic moiety, most preferably the subject moiety is succinimidyl, phthalimidyl or maleimidyl; and, wherein the substituents are selected from those set forth in regard to L, above, but preferably those set forth in regard to V, above, and
(iv) R2, R5, R6, R7, R8, Q and n are selected as set forth above in regard to any of (i)-(iii), above, or (v)-(vii), below;
R3 and R4 are substituted or unsubstituted lower alkylene, preferably C1-4 alkylene, or lower alkylene linked to a heteroatom, preferably C2-4 alkylene linked to a heteroatom, or a heteroatom; together with the atoms to which they are attached, R3 and R4 form a heterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, most preferably morpholino, thiomorpholino, pyrrolidinyl, or V-substituted pyrrolidinyl, particularly 4-hydroxypyrrolidinyl; wherein, the substituents of R3 and/or R4 are selected from those set forth in regard to L, above, but preferably to those set forth in regard to V, above; or
(v) R2, R7, R8, Q and n are selected as set forth in regard to (i), above;
R3 and R4 are selected as in either of (i) or (iv), above;
R5 and R6 are each independently substituted or unsubstituted lower alkylene, preferably C2-4 alkylene, or lower alkylene linked to a heteroatom, preferably C2-4 alkylene linked to a heteroatom, or a heteroatom; together with the atoms to which they are attached R5 and R6 form a heterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, most preferably a moiety selected from among morpholino, thiomorpholino, pyrrolidinyl, or V-substituted pyrrolidinyl, particularly 4-hydroxypyrrolidinyl; wherein, the substituents of R5 and/or R6 are selected from those set forth in regard to L, above, but preferably to those set forth in regard to V, above; or
(vi) R2, R6 and R8 are selected as set forth in regard to (i), above;
R3 and R4 are selected as in either of (i) or (iv), above;
n is zero; and
R5 and R7 are substituted or unsubstituted lower alkylene, preferably C2-4 alkylene, or lower alkylene linked to a heteroatom, preferably C2-4 alkylene linked to a heteroatom, or a heteroatom; together with the atoms to which they are attached R5 and R7 form a heterocyclic moiety, preferably a 4-6 membered heterocyclic moiety, most preferably selected from among morpholino, thiomorpholino, pyrrolidinyl, or V-substituted pyrroliclinyl, particularly 4-hydroxypyrrolidinyl; wherein, the substituents of R5 and/or R7 are selected from those set forth in regard to L, above, but preferably to those set forth in regard to V, above; or
(vii) R2, R5 and R8 are selected as set forth in regard to (i), above;
R3 and R4 are selected as in either of (i) or (iv), above;
n is 0;
R6 and R7 together with the atoms to which each is attached form a 6-12, preferably an 8-10 membered bicyclic heterocyclic or heteroaryl moiety, preferably a reduced isoquinolinyl moiety, more preferably a 1,2,3,4-tetrahydroisoquinolinyl moiety;
and in all of (i)-(vii), above, unless specified otherwise: (a) the carbon chains, where so indicated, are straight or branched chain containing from 1 to about 12 carbons preferably 1 to about 6, and most preferably 4 to about 6 carbons; and (b) the cyclic moieties, where so indicated, contain one ring or two fused rings, each ring, or combination of rings, preferably containing 3 to about 16 atoms, most preferably 4 to about 12 atoms, and in a combination of two fused rings most preferably each of the subject rings contains 4 to about 6 ring atoms.
Abbreviations used are e.g., AD, Alzheimer""s disease; Axcex2, Axcex2-amyloid peptide; APP, amyloid precursor protein; APP751, wild-type human APP-751 protein; APP695WT, wild-type human APP-695 protein; APP670/671, human APP-695 protein harboring the xe2x80x9cSwedishxe2x80x9d double mutation at codons 670 and 671; APP670/671/717, human APP-770 protein containing the xe2x80x9cSwedishxe2x80x9d double mutation at codons 670 and 671 and a Phe for Val substitution at codon 717 (codons numbered according to the APP-770 amino acid isoform); xcex1-sAPP, soluble APP; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FBS, fetal bovine serum; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMSO, dimethylsulfoxide.
The terms used herein are intended to have meaning as follows: namely,
xe2x80x9cAlkylxe2x80x9d is intended to mean straight, branched-chain or cyclic hydrocarbon chain composed of a number of carbon atoms that is commonly specified according to a formula. Where not specified, alkyl groups in the instant compositions preferably contain about 1 to about 12 carbon atoms (C1-12). Representative examples of the subject alkyl groups include methyl, ethyl, propyl, cyclopropyl, isopropyl, n-butyl, t-butyl, sec-butyl, cyclobutyl, pentyl, cyclopentyl, n-hexyl, n-nonyl, n-decyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl, and the like.
xe2x80x9cAlkenylxe2x80x9d is intended to mean a straight, branched-chain or cyclic hydrocarbon chain composed of one or more double-bonded carbon atoms, a number of which carbon atoms is commonly specified according to formula. Where not specified, alkenyl carbon chains in the instant compositions preferably contain about 2 to about 12 carbon atoms (C2-12), and preferably about 2 to about 7 carbon atoms (C2-7). The subject C2-12 alkenyl carbon chains contain about 1 to about 6 double bonds, while C2-7 alkenyl carbon chains preferably contain about 1 to about 3 double bonds. Representative alkenyl moieties include 2-methyl-2-propenyl, 2-methyl-1-propenyl, propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2,2-difluoroethenyl, as well as, those straight and branched chain moieties having up to two double bonds. Cyclic alkenyl carbon moieties preferably contain one or two fused hydrocarbon ring moeities having about 3 to about 16 carbon atoms (C3-16), and most preferably having about 4 to about 12 carbon atoms (C4-12).
xe2x80x9cAlkynylxe2x80x9d is intended to mean a straight, branched-chain or cyclic hydrocarbon chain composed of one or more triple-bonded carbon atoms, a number of which carbon atoms is commonly specified according to formula. Where not specified, alkynyl carbon chains in the instant compositions preferably contain about 2 to about 12 carbon atoms (C2-12) with, preferably, about 1 to about 6 triple bonds, and the alkynyl carbon chains of 2 to 6 carbons (C2-6), preferably, contain 1 to 3 triple bonds. Representative alkynyl moieties include propynyl. Cyclic carbon alkynyl moieties preferably contain one or two fused hydrocarbon ring moeities having about 3 to about 16 carbon atoms (C3-16), and most preferably about 4 to about 12 carbon atoms (C4-12). xe2x80x9cLower alkylxe2x80x9d, xe2x80x9clower alkenylxe2x80x9d and xe2x80x9clower alkynylxe2x80x9d are intended to mean alkyl, alkenyl and alkynyl hydrocarbon chains, respectively (as defined supra), each of which chains contains fewer than six carbon atoms.
xe2x80x9cCycloalkylxe2x80x9d is intended to mean an unsaturated carbon bond single ring system moiety, or an unsaturated carbon bond multiple cyclic ring system moiety. Preferably, the subject cycloalkyl consists of about 3 to about 10 carbon atoms, and most preferably, the subject cycloalkyl consists of about 3 to about 6 carbon atoms. Representative example of the subject ring system moieties include one, two or more rings that are joined together in a fused, bridged or spiro-connected fashion, each of which moieties may be optionally substituted with one or more alkyl group substituents.
xe2x80x9cCycloalkenylxe2x80x9d is intended to mean a single ring system moiety, or a multiple ring system each of which moieties has at least one carbon-carbon double bond, and each of which moieties preferably contains about 3 to about 10 carbon atoms (C3-10), and most preferably, about 4 to about 7 carbon atoms (C4-7). Representative example of the subject ring system moieties include one, two or more rings that are joined together in a fused, bridged or spiro-connected fashion, each of which moieties may be optionally substituted with one or more alkyl group substituents.
xe2x80x9cCycloalkynylxe2x80x9d is intended to mean a single ring system moiety, or a multiple ring system each of which moieties has at least one carbon-carbon triple bond, and each of which moieties preferably contains about 3 to about 10 carbon atoms (C3-10), and most preferably, about 8 to about 10 carbon atoms (C8-10). Representative example of the subject ring system moieties include one, two or more rings that are joined together in a fused, bridged or spiro-connected fashion, each of which moieties may be optionally substituted with one or more alkyl group substituents.
xe2x80x9cHeteroatomsxe2x80x9d is intended to mean that the subject atoms are not the same. Where not specified, heteroatoms in the instant compositions are commonly non-carbon atoms which are preferably are selected from among Oxygen (O), Nitrogen (N) and Sulfur (S). Substituents containing heteroatoms may be straight chain, branched chain or heterocyclic.
xe2x80x9cArylxe2x80x9d is intended to mean an aromatic hydrocarbon cyclic moiety, specified according to formula. Where not specified, in the instant compositions preferably the aryl moieties contain about 3 to about 14 carbon atoms (C3-14) bonded into about 1 to about 2 ring structures. xe2x80x9cLower arylxe2x80x9d is intended to mean that the subject aryl moiety, defined supra, preferably contain about 5 to about 7 carbon atoms in the subject ring structure. xe2x80x9cAralkylxe2x80x9d is intended to mean that the subject aryl moiety, defined supra, is additionally substituted with one or more alkyl groups, as defined supra. Representative examples of aralkyl and aryl moieties include phenyl; benzyl; phenethyl; 1- and 2-naphthylmethyl; 1- and 2-naphthyl; 1- and 2-indenyl; pentalenyl; azulenyl; heptalenyl; acenaphthylenyl; 9-fluorenyl; phenalenyl; phenanthrenyl; anthracenyl; triphenylenyl; pyrenyl, chrysenyl; naphthacenyl; and the like.
xe2x80x9cHeterocyclexe2x80x9d, and xe2x80x9cheterocyclicxe2x80x9d, are intended to mean a cyclic carbon atom ring moiety composed of about 3 to about 14 atoms (C3-14), preferably about 3 atoms to about 7 atoms constituting about 1 to about 2 ring moieties; of which atoms, one or more atoms are heteroatoms, as defined supra, and the subject preferred heteroatoms are as set forth above. Representative heterocycles include aliphatic saturated carbon ring moities bonded with unsaturated heteroaryl ring moieties. Representative examples of heterocycles include pyrrolidinyl, piperidinyl, alkylpiperidinyl, 1,4-dioxanyl, 1,4-dithianyl, 1,4-morpholinyl, 1,4-thiomorpholinyl, 1,2,3-oxadiazolyl, 1,3,5-trithianyl or 1,2,5-triazolyl.
xe2x80x9cHeteroarylxe2x80x9d is intended to mean a cyclic carbon atom ring moiety composed of about 3 to about 14 atoms (C3-14), preferably about 3 atoms to about 7 atoms constituting about 1 to about 2 ring moieties; of which atoms, one or more atoms is a heteroatom, as defined supra. The subject preferred heteroatoms are as set forth above, and most preferably at least one ring moiety comprises an aromatic ring. Representative examples of heteroaryls include furyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, pyranyl, indolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, naphthyridinyl, benzothiazolyl, dibenzothiazolyl, xanthenyl and 1,2,3,4-tetrahydroisoquinolinyl.
xe2x80x9cNRARBxe2x80x9d is intended to mean a chemical moiety in which a nitrogen atom (N) is bonded to both an RA substituent and an RB substituent.
In certain alternative embodiments, one or more R group substituents, e.g., R3, R5, and the like, of Formula I, II or III, supra, may be identical with a side chain residue of a xe2x80x9cnaturally occurring amino acidxe2x80x9d. xe2x80x9cNaturally occurring amino acidsxe2x80x9d, and their constituent xe2x80x9cside chain residuesxe2x80x9d are known in the art. xe2x80x9cNatural side chain residues of naturally occurring amino acidsxe2x80x9d, as used herein, is intended to mean natural substituents attached to the xcex1-carbon atom of a naturally occurring amino acid synthesized by a plant or an animal. Representative examples of natural side chain residues include the hydrogen atom attached as a substituent at the xcex1-carbon in glycine, the methyl group attached at the xcex1-carbon in alanine, the isopropyl group attached at the xcex1-carbon in valine, and the like.
xe2x80x9cHalogenxe2x80x9d, xe2x80x9chalidexe2x80x9d and/or xe2x80x9chaloxe2x80x9d are used interchangeably to refer substituents selected from among a fluorine atom (F), chlorine atom (Cl), bromine atom (Br), iodine atom (I), as well as pseudohalide atoms. Where not specified, halogen in the instant compositions preferably contain one or more atoms selected from among fluorine, chlorine, bromine and iodine.
xe2x80x9cPseudohalidesxe2x80x9d is intended to mean atoms, or chemical constituent groups, that have one or more chemical properties and/or chemical reactivities substantially similar to a halogen. The subject constituent groups are distinguished by being interchangeable with halogens in the instant synthetic methods, as disclosed further below. Representative examples of pseudohalides include, but are not limited to, cyanide, cyanate, thiocyanate, selenocyanate, azide and trifluoromethyl.
xe2x80x9cHaloalkylxe2x80x9d is intended to mean an alkyl group substituted with one or more halogens.
xe2x80x9cHalo-lower alkylxe2x80x9d is intended to mean an alkyl moiety containing six or fewer carbon atoms which is substituted with one or more halogens. Where not explicitly specified otherwise, preferred halo-lower alkyl moieties in the instant compositions are selected from among xe2x80x94CF3, xe2x80x94CF2H, xe2x80x94CFH2, xe2x80x94CH2Cl and xe2x80x94CH2Br.
xe2x80x9cHaloalkoxyxe2x80x9d is intended to mean an RO- moiety in which R is a haloalkyl group.
xe2x80x9cAminocarbonylxe2x80x9d is intended to mean an xe2x80x94C(O)NH2 moiety.
xe2x80x9cAlkylaminocarbonylxe2x80x9d is intended to mean an xe2x80x94C(O)NHR moiety in which R is hydrogen, alkyl, preferably lower alkyl or aryl, preferably lower aryl.
xe2x80x9cDialkylaminocarbonylxe2x80x9d is intended to mean an xe2x80x94C(O)NRxe2x80x2R moiety in which Rxe2x80x2 and R are independently alkyl or aryl, preferably lower alkyl or lower aryl;
xe2x80x9cCarboxamidexe2x80x9d is intended to mean groups of formula xe2x80x94NRxe2x80x2COR.
xe2x80x9cAlkoxycarbonylxe2x80x9d is intended to mean an xe2x80x94C(O)OR group in which R is alkyl, preferably lower alkyl or aryl, preferably lower aryl.
xe2x80x9cAlkoxyxe2x80x9d is intended to mean an ROxe2x80x94 group in which R is alkyl, preferably a lower alkyl or aryl, and most preferably a lower aryl.
xe2x80x9cThioalkoxyxe2x80x9d is intended to mean and RSOxe2x80x94 group in which R is alkyl, preferably a lower alkyl or aryl, and most preferably a lower aryl.
xe2x80x9cAlkylenexe2x80x9d is intended to mean a straight, branched or cyclic hydrocarbon moiety, preferably a straight or branched moiety composed of about 1 to about 20 carbon atoms (C1-20), and most preferably composed of fewer than about 12 carbon atoms (C1-12). In presently preferred embodiments, the alkylene moiety is either unsubstituted, or alternatively, is substituted with one or more alkyl groups, as defined supra. In certain alternative embodiments of the invention, the subject hydrocarbon moiety making up an alkylene additionally contains one or more heteroatoms selected from among oxygen and nitrogen. In other embodiments, the latter subject nitrogen heteroatom can constitute an amide nitrogen bonded with an alkyl group. Representative examples of alkylene groups include methylene (xe2x80x94CH2xe2x80x94), ethylene (xe2x80x94CH2CH2xe2x80x94), propylene (xe2x80x94(CH2)3xe2x80x94), cyclohexylene (xe2x80x94C6H,Oxe2x80x94), methylenedioxy (xe2x80x94Oxe2x80x94CH2xe2x80x94Oxe2x80x94) and ethylenedioxy (xe2x80x94Oxe2x80x94(CH2)2xe2x80x94Oxe2x80x94).
xe2x80x9cLower alkylenexe2x80x9d is intended to mean an alkylene moiety composed of fewer than about 6 carbon atoms. In certain preferred embodiments, lower alkylene groups contain about 2 to about 3 carbon atoms.
xe2x80x9cAlkenylenexe2x80x9d is intended to mean a straight, branched or cyclic hydrocarbon, preferably straight or branched chain composed of about 2 to about 20 carbon atoms (C2-20) at least two of which are double bonded one to another. In certain presently preferred embodiments of the invention, the subject alkenylene moiety is preferably composed of about 2 to about 12 carbon atoms (C2-12), and in most preferred embodiments the subject moiety preferably consists of a xe2x80x9clower alkenylenexe2x80x9d having about 2 to about 6 carbon atoms. In certain alternative embodiments, the subject alkenylene moiety may be composed of a compound substituted at one or more positions in the straight, branched or cyclic hydrocarbon with one or more alkyl groups, as defined supra. In other alternative embodiments, the subject straight, branched or cyclic hydrocarbon may contain one or more heteroatoms, i.e., in place of one or more carbon atoms. In the latter case, the subject heteroatoms are preferably oxygen, sulfur or nitrogen atoms. The latter nitrogen atom may, in other alternative embodiments, constitute an amide nitrogen bonded with an alkyl group. Representative examples of alkenylene groups include xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94 and xe2x80x94CHxe2x95x90CHxe2x80x94CH2xe2x80x94.
xe2x80x9cLower alkenylenexe2x80x9d is intended to mean an alkenylene having about 2 to about 6 carbon atoms, and preferably about 2 to about 4 carbon atoms.
xe2x80x9cAlkynylenexe2x80x9d is intended to mean a straight, branched or cyclic hydrocarbon, preferably straight or branched chain composed of about 2 to about 20 carbon atoms (C2-20) at least two of which are triple bonded one to another. In certain presently preferred embodiments, the subject alkynylene moiety preferably comprises about 2 to about 12 carbon atoms (C2-12), and in most preferred embodiments the subject moiety consists of a xe2x80x9clower alkynylenexe2x80x9d having 2 to about 6 carbon atoms. In certain alternative embodiments, the subject alkynylene moiety may be composed of a compound substituted at one or more positions in the straight, branched or cyclic hydrocarbon with one or more alkyl groups. In other alternative embodiments, the subject straight, branched or cyclic hydrocarbon may contain one or more heteroatoms, i.e., in place of one or more carbon atoms. The latter heteroatoms are preferably oxygen, sulfur or nitrogen atoms. The latter nitrogen may, in other alternative embodiments, constitute an amide nitrogen bonded with an xe2x80x9calkyl group substituentxe2x80x9d, as defined below. Representative examples of alkynylene groups include xe2x80x94Cxe2x89xa1Cxe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94 and xe2x80x94Cxe2x89xa1Cxe2x80x94CH2xe2x80x94.
xe2x80x9cLower alkynylenexe2x80x9d is intended to mean an alkynylene composed of about 2 to about 6 carbons. In presently preferably embodiments of the invention, lower alkynylene moieties are composed of about 2 to about 4 carbon atoms.
xe2x80x9cArylenexe2x80x9d is intended to mean a monocyclic or polycyclic hydrocarbon ring moiety having one or more aromatic/aryl groups as constituents in the subject ring moiety, and additionally, having one or more carbon-carbon double bonds. The arylene moiety is preferably composed of 3 to about 20 carbon atoms (C3-20). In presently most preferred embodiments, the subject arylene groups are composed of about 3 to about 12 carbon atoms (C3-12), and most preferably, the arylene group is a xe2x80x9clower arylenexe2x80x9d (C5-6). In alternative embodiments, the subject arylene moiety may, in turn, be substituted with one or more aryl groups, and the arylene cyclic ring form may also, optionally, contain one or more heteroatoms. The latter heteroatoms constituting the arylene cyclic ring are preferably oxygen, sulfur or nitrogen, and the subject nitrogen atom may, in other embodiments, constitute an amide nitrogen bonded to an xe2x80x9calkyl group substituentxe2x80x9d. Representative examples of arylene groups include 1,2-, 1,3- and 1,4-phenylenediamine.
xe2x80x9cLower arylenexe2x80x9d is intended to mean an arylene group having about 8 to about 10 carbon atoms (C8-10).
xe2x80x9cAlkylidenexe2x80x9d is intended to mean a xe2x95x90CRxe2x80x2Rxe2x80x3 substituent group, in this case, attached to a basal hydrocarbon core structure such as a xe2x95x90CRxe2x80x2Rxe2x80x3 group substituent of an alkylene or a heterocycleene. In the latter case, the xe2x95x90CRxe2x80x2Rxe2x80x3 substituent group is attached to the core structure through the group""s carbon-carbon double bond (xe2x95x90Cxe2x80x94). Representative examples of alkylidene groups include methylidene (xe2x95x90CH2) and ethylidene (xe2x95x90CHCH3).
xe2x80x9cArylalkylidenexe2x80x9d is intended to mean an alkylidene group, i.e., xe2x95x90CRxe2x80x2Rxe2x80x3, in which either the Rxe2x80x2 or the Rxe2x80x3 substituent constitutes an aryl group.
xe2x80x9cCarbonylxe2x80x9d is intended to mean a bivalent carbonyl group of the general formula (xe2x80x94(Cxe2x95x90O)xe2x80x94).
xe2x80x9cAlkyl group substituentxe2x80x9d is intended to mean a hydrocarbon, a halo-hydrocarbon or a halogen-containing substituent to a core structure as follows: namely, in presently preferred embodiments, the subject xe2x80x9calkyl group substituentxe2x80x9d is halo-lower-alkyl, aryl, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, an oxygen atom or a cycloalkyl group.
xe2x80x9cAryl group substituentxe2x80x9d, when used in regard to a core structure that is alkyl, cycloalkyl, cycloalkylalkyl, aryl, or a heteroaryl moiety, is intended to mean that the subject core structure is substituted with 1 or more, and preferably 1 to about 3, xe2x80x9caryl group substituentsxe2x80x9d selected from the following: namely, halogen atoms, alkyls, halo-alkyls, arylalkyl, heteroarylalkyl, alkenylene, alkynylene and hydroxyl. In presently preferred embodiments of the invention wherein the core structure is a halo-lower-alkyl, the preferred xe2x80x9caryl group substituentsxe2x80x9d are trifluoromethyl, formyl, alkylcarbonyl or substituted ore unsubstituted arylcarbonyl. In the latter case the substituents comprise 1 or more, and preferably 1 to about 3 groups selected from among the following: namely, halogen atoms, halo-alkyls, alkyls, heteroarylcarbonyls, carboxyls; alkoxycarbonyls, aryloxycarbonyls, aminocarbonyls, alkylaminocarbonyls, dialkylaminocarbonyls, arylamino-carbonyls, diarylaminocarbonyls, arylalkylaminocarbonyls, alkoxys, aryloxys, perfluoroalkoxys, alkenyloxys, alkynyloxys, arylalkoxys, aminoalkyls, alkylaminoalkyls, dialkylaminoalkyls, arylaminoalkyls, amino groups, alkylamino groups, dialkylamino groups, arylaminos, alkylarylaminos, alkylcarbonylaminos, arylcarbonyls-, amines, azidos, nitrogen atoms, thiol groups, alkylthiol groups, arylthiols, perfluoroalkylthiols, thiocyano groups, isothiocyano groups, alkylsulfinyl groups, alkylsulfonyls, arylsulfinyls, arylsulfonyls, aminosulfonyls, alkylaminosulfonyls, dialkylaminosulfonyls and arylaminosulfonyl groups.
xe2x80x9cAminosulfonylxe2x80x9d is intended to mean either an xe2x80x94NHSO2xe2x80x94 substituent or an xe2x80x94SO2NHxe2x80x94 bivalent group.
xe2x80x9cAmidoxe2x80x9d is intended to mean either a xe2x80x94C(O)NHxe2x80x94 or an xe2x80x94HNC(O)xe2x80x94 bivalent group.
xe2x80x9cThioamidoxe2x80x9d is intended to mean either a xe2x80x94C(S)NHxe2x80x94 or an xe2x80x94HNC(S)xe2x80x94 bivalent group.
xe2x80x9cOxyamidoxe2x80x9d is intended to mean a xe2x80x94OC(O)NHxe2x80x94 or an xe2x80x94HNC(O)Oxe2x80x94 bivalent group.
xe2x80x9cThiaamidoxe2x80x9d is intended to mean a xe2x80x94SC(O)NHxe2x80x94 or an xe2x80x94HNC(O)Sxe2x80x94 bivalent group.
xe2x80x9cDithiaamidoxe2x80x9d is intended to mean a xe2x80x94SC(S)NHxe2x80x94 or an xe2x80x94HNC(S)Sxe2x80x94 bivalent group.
xe2x80x9cUreidoxe2x80x9d is intended to mean an xe2x80x94HNCONHxe2x80x94 bivalent group.
xe2x80x9cThioureidoxe2x80x9d is intended to mean an xe2x80x94HNCSNHxe2x80x94 bivalent group.
xe2x80x9cAlzheimer""s diseasexe2x80x9d is the most common form of dementia affecting nearly 25% of the elderly population over the age of 85 years. Alzheimer""s disease (AD) can now be diagnosed with a considerable degree of accuracy with 90% correspondence between clinical diagnosis and autopsy confirmation at certain centers. The characteristic histopathologic changes at autopsy include neurofibrillary tangles, neuritic xe2x80x9csenilexe2x80x9d plaques, neuronal loss and amyloid angiopathy (1). The major constituent of plaques (plaques) is a 4.2 kDa fibrillogenic polypeptide of 42-43 amino acids in length and referred to as amyloid beta-protein (abbreviated Axcex2). Axcex2 is derived by proteolytic cleavage from amyloid precursor protein (APP; 2-5) and accumulates in the cerebral extracellular perivascular matix in patients with AD, where its observance is considered to be a pathological landmark of the disease. When aggregated in fibrillar beta-pleated sheet configurations, Axcex2 is thought to form a nidus for binding of other proteins that may contribute to pathology. Binding of Axcex2 to neurons can result in increased vulnerability to excitotoxicity, generation of oxygen free radicals, impaired membrane transport systems, inhibition of glutamate receptors and induction of apoptosis (6). Neuronal loss can lead to disruption and defects in neurotransmission, cell-cell communications and abnormal production of extracellular matrix proteins. In addition, deposition of Axcex2 in and around cerebral blood vessels, i.e., cerebral amyloid angiopathy, has been associated with hemorrhagic stroke in the elderly and patients with AD.
xe2x80x9cAmyloid precursor proteinxe2x80x9d. Abbreviated APP, is intended to mean an amino acid sequence of National Library of Medicine Accession No. 1070623, having 695 or 770 amino acids, and sequences related thereto e.g. by conservative amino acid substitution and the like Representative proteins have one or more of the following attributes: namely, a calculated theoretical molecular weight about 86,943 encoded by a gene mapping to 21q21.2 and 2-21q21.2, alternatively spliced to encode either a protein of 695aa or 770aa as a C-terminal extended polypeptide with a Kunitz-type serine protease inhibitor doimain, Gly700-Leu723 being the transmembrane domain, and Lys723-Phe695 or Lys723-Asn770 being the cytoplasmic domain.
xe2x80x9cAxcex2 domainxe2x80x9d, also referred to as Axcex2-peptidexe2x80x9d, and abbreviated xe2x80x9cAxcex2xe2x80x9d, is intended to mean a cleavage product of APP, supra, as mediated by one or more proteases at an APP NH2-terminal Lys670-Met671 or Met671-Asp672 residue and a COOH-terminal Val710-Va711 or at a COOH-terminal Ala713-Thr714. xe2x80x9cAxcex21-40xe2x80x9d being Asp672-Ile712; xe2x80x9cAxcex21-39xe2x80x9d being Asp672-Val711; Axcex21-42 being Asp672-Thr714; and xe2x80x9cAxcex21-43xe2x80x9d being Asp672-Val715.
A xe2x80x9cCarboxy terminal fragmentxe2x80x9d, abbreviated interchangeably CTF and ixcex2-CTF, is intended to mean the portion of APP (Accession No. 1070623) corresponding to an APP fragment having a molecular size on SDS-PAGE of about 9 kDa to about 12 kDa and/or a sequence from about Asp672 to about Asn770.
xe2x80x9cCTF 100xe2x80x9d, meaning carboxy terminal fragment of 100 amino acids in length, is intended to mean an APP fragment having an apparent molecular size on SDS-PAGE of about 9 kDa to about 12 kDa and a sequence from about Asp672 to about Asn770.
xe2x80x9cSoluble APPxe2x80x9d, abbreviated sAPP, is intended to mean the portion of APP (Accession No. 1070623) corresponding to an N-terminal fragment produced by xcex1-secretase having a molecular size on SDS-PAGE of about 100 kDa to about 115 kDa and lacking the transmembrane (supra) and cytoplasmic (supra) domains of APP.
xe2x80x9cTreatmentxe2x80x9d is intended to mean a method of delivering to a subject in need thereof a pharmaceutical preparation according to the compounds of the invention with the aim of effecting a change in APP:Axcex2 processing in the subject in need thereof. The instant methods include delivering the preparation to a patient i) before the dysfunction has been diagnosed, e.g., prophylactic protocols delivered with the aim of preventing development of the dysfunction, as well as, ii) after the dysfunction has been diagnosed, e.g., therapeutic protocols. That the subject treatments have fulfilled the intended aim of effecting a change in APP processing to Axcex2 in the subject will be evident by one or more of the following: namely, (i) a change in the amount of Axcex2 or sAPP in a sample of a biological tissue or biological fluid collected from a treated patient; (ii) a change in the amount of one or more APP:Axcex2 processing proteins in a biological tissue or biological fluid collected from a treated patient; or, (iii) a change (increase or decrease) or complete elimination of one or more clinical indicia of disease, e.g., diagnostic symptoms, in a treated patient.
xe2x80x9cDysfunctionxe2x80x9d is intended to mean a pathologic condition of APP processing to Axcex2 in a tissue, i.e., one or more changes in APP processing and/or production of Axcex2 as compared with the processing of APP occurring in a normal healthy control cohort. For example the subject pathological conditions include, but are not limited to, i) toxic amyloid dystrophy, (e.g., chemical or drug-induced secondary amyloid dystrophy), ii) vascular impairment with amyloid deposition, iii) degeneration and peripheral degeneration of vascular and/or neural tissue associated with amyloid deposition, iv) cellular detachment from basement membrane and defects in tissue organization visible microscopically in histology sections, e.g., in the endothelium of vessels; v) amyloid containing lesions resulting following physical trauma; and v) hereditary amyloid deposition in tissues, e.g., systemic amyloidosis and familial Alzheimer""s diseases.
xe2x80x9cSubject in need thereofxe2x80x9d is intended to mean a mammal, e.g., humans, domestic animals and livestock, having one or more dysfunctions of APP processing, as defined above.
xe2x80x9cBiological fluidxe2x80x9d as used herein is intended to mean a tissue fluid, such as cerebrospinal fluid (CSF); blood, plasma and serum; fluid collected from a body cavity (i.e., peritoneal fluid, lung lavage fluid, urogenital mucus secretions, and the like); urine, feces, sputum, sweat; and the like.
xe2x80x9cProcessing of APP to Axcex2xe2x80x9d, abbreviated APP:Axcex2 processing, is intended to mean intracellular pathways by which proteases act upon APP to catalyze hydrolysis of peptide bonds thereby liberating the Axcex2 fragment (as defined supra) of APP; in this case, including processes active in the following intracellular compartments: namely, in the endoplasmic reticulum; in the cytoplasm associated with ribosomes; in cytoplasmic vesicles (e.g., Golgi secretory vesicles); and, in lysosomal vesicles and the like.
xe2x80x9cSubstantially purexe2x80x9d is intended to mean that the subject preparation is sufficiently homogeneous to appear free of detectable impurities as determined by standard methods of physicochemical analysis, e.g. IR, NMR, spectral methods for determining chirality and the like, or by separation methods, e.g., thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas liquid chromatography (GLC) such as subject to analysis by mass spectrometry (GCMS), or by another method employed to assess chemical purity. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification may increase the specific activity of the compound e.g. by isolating a single isostere.
xe2x80x9cProdrugxe2x80x9d is intended to mean one or more of the instant compounds in a form that, upon in vivo administration, is metabolized and/or otherwise converted from a form lacking in a biological or pharmaceutical activity, into a form having the subject activity. Representative examples of forms of prodrugs are disclosed e.g. in Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, at pages 388-392.
xe2x80x9cIsostereoisomerxe2x80x9d, abbreviated isostere, is intended to mean of a compound possessing of one of two or more stereochemical attributes expressed by one of the instant compounds, e.g. one spectral property. An isostere compound may exhibit a biologic and/or a pharmacologic activity that is greater than, or less than that of a racemic mixture of one of the instant compounds.
xe2x80x9cAlter APP processing to Axcex2xe2x80x9d, is intended to mean altering the processing of APP. Two alternative processing pathways for APP may be represented as follows (the arrows represent cleavage sites): 
xe2x80x9cAltering processing of APP to Axcex2xe2x80x9d, abbreviated altering APP:Axcex2 processing, when used in regard to a composition containing one or more of the instant compounds is intended to mean that the subject composition is effective when administered to a cell, e.g., in tissue culture, to inhibit the production of carboxy terminal fragments of APP including ixcex2-CTF and/or Axcex2. While not wishing to be tied to any particular mechanism of action, representative examples of mechanisms by which the latter inhibitory effect on Axcex2 production may be exerted by the instant compounds include inhibiting: (a) an enzyme activity in a cell, e.g., an enzyme exhibiting a xcex3-secretase activity; (b) a co-factor binding to, and exerting an effect on, the subject enzyme activity in the cell, e.g., a co-factor increasing the catalytic efficiency or turnover rate of an enzyme with, a xcex3-secretase activity in a cell; (c) a transcription regulator, e.g., altering the level of expression of mRNA encoding the subject enzyme or cofactor protein in a cell; (d) a protein kinase, e.g., altering either the level of expression of, or the activity of, the subject enzyme in a cell; and (e) a phosphorylase, e.g., altering the activity of the subject enzyme in the cell. The subject compositions may act directly on enzyme:cofactor:APP substrate complexes, or they may act indirectly, i.e., to change transcription or translation of enzyme proteins or cofactors.
xe2x80x9cAltering apoptosisxe2x80x9d is intended to mean that the subject compound is effective to inhibit a cysteinyl protease activated in a cell which has been treated to induce programmed cell death. Representative proteases so activated include calpains, caspases (e.g., caspases 3, 8 and 9), cathepsins and the like. Representative apoptosis inducer agents so able to induce apoptosis include Fas antibody, staurosporine, interleukins IL-1 and TNF, glucose and oxygen deprivation during ischemic tissue injury and the like. Apoptosis may be induced in the cells of tissues subject to traumatic injury, restricted blood flow (e.g., during peripheral microvascular clotting and stroke) and the like.
xe2x80x9cApoptosisxe2x80x9d is intended to mean the cascade of energy (ATP) dependent events triggered by an apoptosis inducer agent and leading to programmed cell death through mechanisms commonly involving intracellular caspase enzymes, and commonly requiring about 12 to about 24 hours to accomplish the subject cell death. Apoptosis does not involve rapid, non-energy dependent lysis of cells, such as occurs during necrosis with release of cellular constituents (e.g., LDH) into the extracellular space. However, during apoptosis cells fragment their DNA and package it into membrane vesicles.
Embodiments of the invention provide presently preferred compounds of FORMULA I, II and III as set forth according to FORMULAS IV, V and VI: as follows, namely, 
wherein, all of the groups and substituents thereof, e.g. R1-R8, Q, Y and Z are identical with those set forth in regard to FORMULA I, II and III, above. In certain alternative preferred embodiments of compounds according to FORMULA IV, V or VI, when (Q)n is a carbonyl group and R1 and R7 and the atoms to which they are attached form a heterocyclic ring moiety, the presently preferred ring moieties are succinimide, phthalimide or maleimide, and the most preferred is phthalimide. Similarly, when n is zero and R1, R7 and the atoms to which they are attached form a heterocyclic ring moiety, the preferred heterocyclic ring moieties are morpholino, thiomorpholino, pyrrolidinyl and V-substituted pyrrolidinyl, and most preferably 4-hydroxypyrrolidinyl and 1,2,3,4-tetrahydroisoquinolinyl. When n is zero and R3, R1 or R2, and R7 taken together with the atoms to which they are attached form heterocyclic moieties, then the preferred ring moieties are morpholino, thiomorpholino, pyrrolidinyl, and V-substituted pyrrolidinyl, and most preferred is 4-hydroxypyrrolidinyl. Representative examples of the presently preferred compounds include: e.g., N-Cbz-L-Leu-L-Leu-L-Leuene-phenyl vinyl sulfone; N-Cbz-L-Leu-L-Leu-D-Leuene-phenyl vinyl sulfone; N-(THIQ-carbonyl)-L-Leu-L-Leu-D-Leuene phenyl vinyl sulfone; N-(THIQ-carbonyl)-L-Leu-L-Leu-L-Leuene phenyl vinyl sulfone, N-(THIQ-carbonyl)-L-Val-L-Met-L-Leuene phenyl vinyl sulfone; N-(THIQ-carbonyl)-L-Val-L-Met-D-Leuene phenyl vinyl sulfone; N-(THIQ-carbonyl)-L-Val-L-Leu-L-Leuene phenyl vinyl sulfone; N-(THIQ-carbonyl)-L-Val-L-Leu-D-Leuene phenyl vinyl sulfone; N-(4-benzylpiperidinyl-carbonyl)-L-Leu-L-Leu-L-Leuene phenyl vinyl sulfone; N-(4-benzylpiperidinyl-carbonyl)-L-Leu-L-Leu-D-Leuene phenyl vinyl sulfone; N-(4-benzylpiperazinyl-carbonyl)-L-Leu-L-Leu-L-Leuene phenyl vinyl sulfone; and, N-(4-benzylpiperazinyl-carbonyl)-L-Leu-L-Leu-D-Leuene phenyl vinyl sulfone.
Embodiments of the invention provide pharmaceutical compositions in which the instant compounds are present as the salts of organic acids and bases (such as acetates, lactates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, fumarates and the like); salts of inorganic acids and bases, e.g., alkali metal salts of lithium, potassium, sodium, alkaline earth metals, such as barium, calcium and magnessium and the like, salts of transition metals such as Zinc, salts of mineral acids such as hydrochlorides and sulfates; esters; acids; bases; solvates; hydrates; amine salts; phosphonates such as sodium hydrogen phosphate and disodium phosphate, and the like; including hydrolyzable prodrugs cleavable and activated in vivo to drugs, e.g., derivatives of N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1xe2x80x2-yl-methylbenzimidazole, diethyl amine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane and the like.
In other embodiments the invention provides a pharmaceutical preparation consisting of one or more isolated and substantially pure diastereoisomers or chiral forms of a pharmaceutical preparations according to FORMULA (I), (II) and/or (III).
Embodiments of the invention provide compounds according to FORMULA I, II or III that are effective to modulate the activity of cysteinyl proteases including extracellular (secreted) proteases and intracellular protease involved in protein processing. The instant protease inhibitors include inhibitors acting, e.g., by processes including both competitive and non-competitive inhibition.
In yet other embodiments, the invention provides methods for administering to a subject in need thereof a concentration of one or more pharmaceutical preparations consisting of one or more compound according to FORMULA I, II and/or III that is effective to ameliorate one or more symptoms of disease. Examples of subjects so-in-need include e.g. patients with systemic amyloidosis, Alzheimer""s disease, atherosclerosis, restenosis following angioplasty, rheumatoid arthritis and connective tissue diseases, dermatologic diseases such as Epidermolysis bullosis, Systemic lupus erythematosus, Sjogren""s syndrome, Insulin-dependent diabetes types I and II, atherosclerosis, stroke, traumatic neural degeneration, chronic vascular and respiratory disorders, necrotic and apoptotic tissue damage resulting from bacterial, fungal and viral infection, (e.g., AIDS dementia, adenovirus, HSV, HPV, influenza and the like), inflammatory bowel disease, irritable bowel syndrome, diarrhea, constipation, gastric acid secretion and ulcers, complications resulting from co-medication or surgical procedures; or heart insufficiency, hyperprolactinemia, asthma and arthritis.
Embodiments also provide compounds according to FORMULA I, II or III, that are capable of inhibiting the activity of a cysteinyl protease, e.g., an intracellular secretase such as xcex2-secretase involved in processing of APP and production of Axcex2, an intracellular apopain/caspase protease involved in programmed cell death (apoptosis), an intracellular protease involved in antigen processing, e.g. a cathepsin, or an extracellular protease involved in tissue destructive processes, e.g., in trauma or arthritis. That a compound is so active may be determined by conducting e.g. either an in vitro or an in vivo assay. For example, in an in vitro assay the instant compounds decrease in the activity of a cellular cysteinyl protease enzyme activity, or a secreted extracellular cysteinyl protease. Assays for cysteinyl proteases, as with assays for other proteases, are within the ordinary skill in the art. In one representative example, the subject cysteinyl protease is an intracellular xcex2-secretase enzyme involved in processing of APP and production of Axcex2 amyloidogenic polypeptides. In the latter case a positive assay is recorded when one or more of the following are observed: namely, (1) a decrease is observed in the 4-kDa amyloid Axcex2 in the medium relative to control cultures; and/or (2) the relative amount of sAPP, in the medium is observed to increase; (3) a decrease is observed in the amount of C-terminal APP fragments  greater than 9 kDa in cell lysates, i.e., resulting from differential processing; and/or (4) there is an observed increase in the amount of xcex1-sAPP in the medium relative to control cultures. In vivo, the instant compounds are preferably effective when administered to an experimental animal of Axcex2 or systemic amyloidosis to either: (1) increase the levels of sAPP (e.g., in brain, plasma or CSF), and/or (2) decrease the level Axcex2 in a biological fluid or tissue extract. In one representative example, a positive assay result may be indicated as an increase in the ratio of xcex1-sAPP to sAPP in a tissue sample from a pre-treatment to a post-treatment interval.
Representative methods for preparing the instant vinyl sulfones are depicted in the EXAMPLES, section below, and in particular in Reaction Schemes A-F of EXAMPLES 1-14. In Scheme A, preparation of amino ester hydrochlorides is depicted; Scheme B illustrates preparation of vinyl sulfones from the amino ester hydrochlorides; in Scheme C, formation of di- and tri-peptide vinyl sulfone analogs is depicted; Scheme D illustrates the preparation of an illustrative isomer of an amino vinyl sulfone; and, synthesis of illustrative N-protected tripeptide vinyl sulfone analogs as 1,2,3,4-tetrahydro-isoquinolinyl ureas is shown in Scheme E. An alternative to the synthesis of Scheme E is presented in Scheme F, i.e., 4-benzylpiperidine or 1-benzylpiperazine are substituted for 1,2,3,4,5-tetra-hydroisoquinoline. As an alternative, an automated peptide synthesizer and solid phase sequential coupling procedures may be used to produce intermediates useful in final synthesis of the instant di- and tri-peptide inhibitors. For example, the instant compounds may be synthesized from their carboxyl terminal end toward their amino terminal end using sequentially protected amino acids, amino acid derivatives and/or isomers, or spacer groups, according to the structure set forth in FORMULAS I, II and/or III. A variety of solid phase resin supports are useful in this method including certain of those useful in the art for solid phase synthesis of peptides. One presently preferred resin comprises polystyrene cross-linked with from about 0.5% to about 3% modified divinyl benzene, wherein the modification involves benzhydrylamidation, chloromethylation or hydroxymethylation. Modification is effective to produce sites that are useful for linking the instant di- and tri-peptide intermediates, e.g., through amide or ester bonds. One representative example of a hydroxymethyl resin is disclosed by Bodansky et al. W 966) Chem. Ind. (London) 38:1597-98). Preparation of illustrative chloromethyl- and benzhydrylamine-resins is disclosed in Stewart et al. (xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d 2nd Edition, Pierce Chemical Co., Rockford, Ill. (1984), Chapter 2, pp. 54-55). Illustrative methods for coupling protected amino acids to a resin are disclosed by Gisin ((1973) Helv. Chem. Acta 56:1476).
A variety of xcex1-amino protecting groups may be useful in synthesis of the instant compounds and their intermediates. Representative examples of such protecting groups as presently contemplated include: (i) acyl protecting groups such as formyl, trifluoroacetyl, benzoyl, phthalyl, p-toluenesulfonyl (tosyl), benzenesulfonyl, nitrophenylsulfonyl, tritylsulfonyl, o-nitrophenoxyacetyl, xcex1-chlorobutyrl, 4-((4-chloro-phenyl)sulfonylaminocarbonyl)phenylcarbonyl, and 4-((4-bromophenyl)sulfonylaminocarbonyl)phenylcarbonyl; (ii) aromatic urethane protecting groups such as phenyloxycarbonyl, benzyloxycarbonyl and substituted benzyloxycarbonyls such as p-chlorobenzy[oxycarbonyl, p-methoxybenzy-[oxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 1-(p-biphenyl)-1-methyl-ethoxycarbonyl, xcex1,xcex1-dimethyl-3,5-dimethoxybenzylo-xycarbonyl, and benzhydryloxycarbonyl; (iii) aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxy-carbonyl, methoxycarbonyl, ethoxycarbonyl, isobutyloxycarbonyl and allyloxycarbonyl; (iv) cycloalkyl urethane protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; (v) thiourethane protecting groups such as phenylthiocarbonyl; (vi) alkyl type protecting groups such as triphenylmethyl (trityl) and benzyl (Bn); and (vii) alkyl and aryl silyl protecting groups such as trimethylsilyl, tert-butyidimethylsilyl, tert-butyl-diphenylsilyl and triisopropylsilyl. t-butyloxycarbonyl (Boc) is one presently preferred xcex1-amino protecting group. Representative methods using Boc for protecting are disclosed by Bodansky et al. in xe2x80x9cThe Practice of Peptide Synthesis,xe2x80x9d Springer-Verlag, Berlin (1984), p. 20). The selection and use of an appropriate coupling reagent is within the skill in the art. Particularly suitable coupling reagents where the amino acid to be added is Gln, Asn, or Arg include N,N-dicyclohexyl-carbodiimide and 1-hydroxybenzotriazole. The use of the latter reagents during synthesis of the instant compounds of FORMULA I, II and/or III prevents nitrile and lactam formation. Other suitable coupling agents include e.g., (i) carbodiimides such as 1-(3-dimethylamino-propyl)-3-tehyl-carbodiimide hydrochloride); (ii) ketenimines; (iii) isoxazolium salts such as N-ethyl-5-phenyl-isoxazolium-3-sulfonate; (iv) monocyclic nitrogen-containing heterocyclic amides of aromatic character containing 1-4 ring nitrogen atoms, e.g., imidazolides, pyrazolides and 1,2,4-triazolides, and particularly N,N-carbonyldiimidazole and N,N-carbonyl-di-1,2,4-triazole; (v) alkoxylated acetylene e.g., ethoxyacetylene; (vi) reagents which form a mixed anhydride with the carboxyl moiety of the amino acid, e.g., ethyl chloroformate and isobutyl chloroformate, or alternatively, the symmetrical anhydride of an amino acid to be coupled e.g., Boc-Ala-O-Ala-Boc; and (vii) nitrogen-containing heterocyclic compounds having a hydroxyl group on one ring nitrogen, e.g., N-hydroxyphthalimide, N-hydroxysuccinimide and 1-hydroxybenzotriazole. Activating reagents, and methods of their use in peptide coupling, are known in the art, e.g. as disclosed in Kapoor ((1970) J. Pharm. Sci. 59:1-27). For synthesis of the instant compounds according to FORMULAS I, II and/or III, presently preferred coupling agents are symmetrical anhydrides. For removal of a compound from a preferred benzhydrylamine resin, treatment with a solution of dimethyl sulfide, p-cresol, thiocresol, or anisole in anhydrous hydrogen fluoride is presently preferred. The cleavage reaction is also preferably carried out at a temperature between about 0xc2x0 C. and about room temperature, over a period of about 5 minutes to about 5 hours.
Embodiments of the invention provide pharmaceutical compositions comprising one or more compounds according to FORMULA I, II and/or III, above. Preferably, each of the instant compounds present in the pharmaceutical composition is present in a therapeutically effective amount. The instant pharmaceutical compositions find a variety of uses in treatments for neurological disorders, e.g., schizophrenia, Alzheimer""s disease, disorders of extrapyramidal motor function, such as Parkinson""s disease, progressive supramuscular palsy, Huntington""s disease, Gilles de la Tourette syndrome and tardive dyskinesia, obesity, severe pain, drug and tobacco withdrawal, respiration, mood and emotional disorders such as depression, anxiety and psychosis, motor control and function, focus and attention disorders, concentration disorders, memory loss, cognitive impairment, dementia (including AIDS dementia), neurodegenerative disorders, epilepsy; as well as in treatments of other diseases, e.g., cardiovascular dysfunction including hypertension and cardiac arrhythmias, convulsive disorders, eating disorders, including bulimia and anorexia, autonomic disorders including dysfunction of gastrointestinal motility and function such as inflammatory bowel disease, irritable bowel syndrome, diarrhea, constipation, gastric acid secretion and ulcers. The instant pharmaceutical compositions may also be useful as co-medication in surgical procedures and pheochromocytoma; or in co-treatments of heart insufficiency, hyperprolactinernia, bacterial infections, asthma or arthritis.
The instant pharmaceutical compositions are preferably formulated as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs. As such the compositions are useful for oral administration, or in sterile solutions or suspensions, for parenteral administration. Transdermal delivery, e.g., patch preparations, and dry powder inhalers are also contemplated. Examples of methods that may be useful for formulating the instant pharmaceutical compositions are disclosed e.g., by Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, p. 126. The instant pharmaceutical compositions may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. Liposome delivery systems are also contemplated, e.g., as disclosed in U.S. Pat. No. 4,522,811.
The concentration of active compound(s) in the instant pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound(s) (i.e., the selected compounds of FORMULA I, II and/or III, above), the physicochemical characteristics of the compound(s), the dosage schedule, and amount administered as well as other factors known to skilled artisans. Typically a therapeutically effective dosage will produce a serum concentration of one or more of the instant compounds ranging from about 0.1 ng/ml to about 50-100 xcexcg/ml serum or plasma. The pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilogram (kg) of body weight per day. Pharmaceutical dosage unit forms may be prepared to provide about 1 mg to about 1000 mg, preferably about 10 mg to about 500 mg of the instant compound. The dosage and method of treatment will be determined by the physician after consideration of the general health, size and condition of a patient.
Effective concentrations (or amounts) of one or more of the compounds described herein or pharmaceutically acceptable derivatives thereof may be mixed with one or more suitable pharmaceutical carriers or vehicles. Solutions or suspensions useful for parenteral, intradermal, subcutaneous, or topical administration may include a sterile diluent such as water for injection, saline solution; a fixed oil; a polyethylene glycol; a glycerin; a propylene glycol or other synthetic solvent; one or more antimicrobial agents, e.g. benzyl alcohol and methyl parabens; an antioxidant, e.g. ascorbic acid or sodium bisulfite; a chelating agent, e.g. ethylenediaminetetraacetic acid (EDTA); a buffer, e.g. an acetate, citrate or phosphate buffer; or, an isotonic agent e.g., sodium chloride or dextrose. Parenteral preparations may be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable materials.
Methods for improving the solubility of the instant compounds, e.g., during administration and delivery from within the instant pharmaceutical compositions, include but are not limited to, uses of co-solvents, e.g., dimethylsulfoxide (DMSO), surfactants, e.g., TWEEN, dissolution in aqueous sodium bicarbonate, and preparation of soluble derivatives or prodrugs of the instant compounds prior to formulation.
Unit dosage forms suitable for administration to subjects in need thereof, e.g., as packaged individually, are known in the art. Each unit-dose commonly contains a predetermined quantity of the therapeutically active compound sufficient to produce a desired therapeutic effect; in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms may be administered in fractions or multiples thereof. Representative methods for preparing dosage forms are known in the art, e.g., see Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.
Oral pharmaceutical dosage forms including solid, gel and liquid are contemplated formed as tablets, capsules, granules, bulk powders, chewable lozenges enteric-coated capsules, sugar-coated or film-coated capsules, hard or soft gelatin capsules, granules e.g., as non-effervescent or effervescent forms, pills, troches and the like. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
Parenteral injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Typically a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, preferably more than 1% w/w of the active compound to the treated tissue(s).
Topical solutions, suspension, emulsions and the like may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, irrigations, sprays, nasal sprays, suppositories, bandages, dermal patches, transdermal delivery systems and the like. Contemplated formulations include inhalent aerosols in the form of an aerosol or nebulizer solutions, inhalent therapy solutions, microfine powders for insufflation, and the like. Also contemplated are formulations for skin and mucous membranes, ophthalmic uses (e.g., eyes), intra-cisternal and intra-spinal application.
The pharmaceutical compositions containing compounds or pharmaceutically acceptable derivatives may be packaged as articles of manufacture containing e.g., a packaging material; the instant pharmaceutical composition; a product insert describing that the composition is effective in ameliorating one or more symptoms of disease, i.e., as set forth above; and, a label indicating that the composition containing the compound or derivative thereof is useful in the treatment or prevention of the disease, i.e., as set forth above.
Embodiments of the invention provide compounds according to FORMULA I, II and/or III that find uses in pharmaceutical compositions manufactured for the purpose of administering a treatment effective in altering apoptosis and altering processing of APP to Axcex2, e.g., altering deposition of amyloid. The dose ranges, which can be established empirically, for use in the treatment of disease states will depend upon the etiology, nature, and severity of the disease state as well as such other factors as determined by the attending physician. The broad range for effective treatment is about 0.01 to 10 mg per kilogram (kg) of body weight per day. The preferred range is about 0.1 to 10 mg/kg of body weight per day. Preferred modes of administration include oral and parenteral modes of administration. It is believed that the instant treatments for patients with such amyloid disorders will effect beneficial changes in the disease process resulting in a stabilization of disease activity, slowed progression of disease, and/or an enhanced lifestyle with increased cognitive ability and decreased anxiety, as well as a possible delay (or obviation) of the need to institutionalize certain patients.
In other embodiments, pharmaceutical compositions are provided comprising the instant compounds of FORMULA I, II and/or III as formulated for use to alter apoptosis, e.g., by inhibiting the action of cysteinyl caspase proteases and/or cathepsin proteases. Ca2+ dependent proteases are presently thought to play significant roles in the pathology of a variety of disorders, including cerebral ischaemia, cataract, myocardial ischaernia, muscular dystrophy and platelet aggregation.